Influence of different functional elements of plasmid pGT232 on maintenance of recombinant plasmids in Lactobacillus reuteri populations in vitro and in vivo

Plasmid pGT232 (5.1 kb), an indigenous plasmid of Lactobacillus reuteri 100-23, was determined, on the basis of nucleotide and deduced protein sequence data, to belong to the pC194-pUB110 family of plasmids that replicate via the rolling-circle mechanism. The minimal replicon of pGT232 was located on a 1.7-kb sequence consisting of a double-strand origin of replication and a gene encoding the replication initiation protein, repA. An erythromycin-selectable recombinant plasmid containing this minimal replicon was stably maintained (>97% erythromycin-resistant cells) without antibiotic selection in an L. reuteri population under laboratory growth conditions but was poorly maintained (90% resistant cells) of pGT232-derived plasmids in the lactobacillus population in vivo required an additional 1.0-kb sequence which contained a putative single-strand replication origin (SSO). The SSO of pGT232 is believed to be novel and functions in an orientation-specific manner

Medical mycology and fungal immunology : new research perspectives addressing a major world health challenge

N.A.R.G. is supported by grants from The Wellcome Trust and MRC. M.G.N. is supported by an ERC consolidator grant (no. 310372).Peer reviewedPublisher PD

Innate immunity glycoprotein gp-340 variants may modulate human susceptibility to dental caries

<p>Abstract</p> <p>Background</p> <p>Bacterial adhesion is an important determinant of colonization and infection, including dental caries. The salivary scavenger receptor cysteine-rich glycoprotein gp-340, which mediates adhesion of <it>Streptococcus mutans </it>(implicated in caries), harbours three major size variants, designated gp-340 I to III, each specific to an individual saliva. Here we have examined the association of the gp-340 I to III polymorphisms with caries experience and adhesion of <it>S. mutans</it>.</p> <p>Methods</p> <p>A case-referent study was performed in 12-year-old Swedish children with high (n = 19) or low (n = 19) caries experiences. We measured the gp-340 I to III saliva phenotypes and correlated those with multiple outcome measures for caries experience and saliva adhesion of <it>S. mutans </it>using the partial least squares (PLS) multivariate projection technique. In addition, we used traditional statistics and 2-year caries increment to verify the established PLS associations, and bacterial adhesion to purified gp-340 I to III proteins to support possible mechanisms.</p> <p>Results</p> <p>All except one subject were typed as gp-340 I to III (10, 23 and 4, respectively). The gp-340 I phenotype correlated positively with caries experience (VIP = 1.37) and saliva adhesion of <it>S. mutans </it>Ingbritt (VIP = 1.47). The gp-340 II and III phenotypes tended to behave in the opposite way. Moreover, the gp-340 I phenotype tended to show an increased 2-year caries increment compared to phenotypes II/III. Purified gp-340 I protein mediated markedly higher adhesion of <it>S. mutans </it>strains Ingbritt and NG8 and <it>Lactococcus lactis </it>expressing AgI/II adhesins (SpaP or PAc) compared to gp-340 II and III proteins. In addition, the gp-340 I protein appeared over represented in subjects positive for Db, an allelic acidic PRP variant associated with caries, and subjects positive for both gp-340 I and Db tended to experience more caries than those negative for both proteins.</p> <p>Conclusion</p> <p>Gp-340 I behaves as a caries susceptibility protein.</p

In Vitro Evaluation of Enterococcus faecalis Adhesion on Various Endodontic Medicaments

E. faecalis in endodontic infection represents a biofilm type of disease, which explains the bacteria’s resistance to various antimicrobial compounds and the subsequent failure after endodontic treatment. The purpose of this study was to compare antimicrobial activities and bacteria kinetic adhesion in vitro for three endodontic medicaments with a clinical isolate of E. faecalis. We devised a shake culture which contained the following intracanalar preparations: CPD, Endoidrox (EIX), PulpCanalSealer (PCS); these were immersed in a liquid culture medium inoculated with the microorganism. The shake system velocity was able to prevent non-specific bacteria adhesion and simulated the salivary flow. Specimens were collected daily (from both the medium and medicaments) for 10 days; the viable cells were counted by plate count, while the adhesion index AI° [E. faecalis fg DNA] /mm2 was evaluated in the pastes after DNA extraction, by quantitative real time PCR for the 16S rRNA gene. A partial growth inhibition, during the first 24 hours, was observed in the liquid medium and on the medicaments for EIX and subsequently for CPD (six logs). EIX showed the lowest adhesion coefficient (5*102 [fg DNA]/mm2) for nine days and was similar to the control. PCS showed no antimicrobial/antibiofilm properties. This showed that “calcium oxide” base compounds could be active against biofilm progression and at least in the short term (2-4 days) on E. faecalis cells growing in planktonic cultures

Changes in the gastric enteric nervous system and muscle: A case report on two patients with diabetic gastroparesis

<p>Abstract</p> <p>Background</p> <p>The pathophysiological basis of diabetic gastroparesis is poorly understood, in large part due to the almost complete lack of data on neuropathological and molecular changes in the stomachs of patients. Experimental models indicate various lesions affecting the vagus, muscle, enteric neurons, interstitial cells of Cajal (ICC) or other cellular components. The aim of this study was to use modern analytical methods to determine morphological and molecular changes in the gastric wall in patients with diabetic gastroparesis.</p> <p>Methods</p> <p>Full thickness gastric biopsies were obtained laparoscopically from two gastroparetic patients undergoing surgical intervention and from disease-free areas of control subjects undergoing other forms of gastric surgery. Samples were processed for histological and immunohistochemical examination.</p> <p>Results</p> <p>Although both patients had severe refractory symptoms with malnutrition, requiring the placement of a gastric stimulator, one of them had no significant abnormalities as compared with controls. This patient had an abrupt onset of symptoms with a relatively short duration of diabetes that was well controlled. By contrast, the other patient had long standing brittle and poorly controlled diabetes with numerous episodes of diabetic ketoacidosis and frequent hypoglycemic episodes. Histological examination in this patient revealed increased fibrosis in the muscle layers as well as significantly fewer nerve fibers and myenteric neurons as assessed by PGP9.5 staining. Further, significant reduction was seen in staining for neuronal nitric oxide synthase, heme oxygenase-2, tyrosine hydroxylase as well as for c-KIT.</p> <p>Conclusion</p> <p>We conclude that poor metabolic control is associated with significant pathological changes in the gastric wall that affect all major components including muscle, neurons and ICC. Severe symptoms can occur in the absence of these changes, however and may reflect vagal, central or hormonal influences. Gastroparesis is therefore likely to be a heterogeneous disorder. Careful molecular and pathological analysis may allow more precise phenotypic differentiation and shed insight into the underlying mechanisms as well as identify novel therapeutic targets.</p

Characterization of the Oral Fungal Microbiome (Mycobiome) in Healthy Individuals

The oral microbiome–organisms residing in the oral cavity and their collective genome–are critical components of health and disease. The fungal component of the oral microbiota has not been characterized. In this study, we used a novel multitag pyrosequencing approach to characterize fungi present in the oral cavity of 20 healthy individuals, using the pan-fungal internal transcribed spacer (ITS) primers. Our results revealed the “basal” oral mycobiome profile of the enrolled individuals, and showed that across all the samples studied, the oral cavity contained 74 culturable and 11 non-culturable fungal genera. Among these genera, 39 were present in only one person, 16 genera were present in two participants, and 5 genera were present in three people, while 15 genera (including non-culturable organisms) were present in ≥4 (20%) participants. Candida species were the most frequent (isolated from 75% of participants), followed by Cladosporium (65%), Aureobasidium, Saccharomycetales (50% for both), Aspergillus (35%), Fusarium (30%), and Cryptococcus (20%). Four of these predominant genera are known to be pathogenic in humans. The low-abundance genera may represent environmental fungi present in the oral cavity and could simply be spores inhaled from the air or material ingested with food. Among the culturable genera, 61 were represented by one species each, while 13 genera comprised between 2 and 6 different species; the total number of species identified were 101. The number of species in the oral cavity of each individual ranged between 9 and 23. Principal component (PCO) analysis of the obtained data set followed by sample clustering and UniFrac analysis revealed that White males and Asian males clustered differently from each other, whereas both Asian and White females clustered together. This is the first study that identified the “basal mycobiome” of healthy individuals, and provides the basis for a detailed characterization of the oral mycobiome in health and disease

Pesan Moral Islami Dalam Film Le Grand Voyage Karya Ismael Ferroukhi: Sebuah Tinjauan Struktural

Kata kunci : Film, sastra, struktural, pesan moral, Islam.Film merupakan produk budaya yang tidak hanya menjadi hiburan di masyarakat, tetapi juga sebagai sarana penyampaian pesan moral yang mengarifkan. Salah satufilm Perancis yang dianggap menginspirasi adalah film berjudul Le grand voyageyang ditulis dan disutradarai oleh Ismaël Ferroukhi. Film ini bercerita tentang perjalanan seorang muslim keturunan Maroko dan anaknya yang bernama Reda.Mereka menempuh jarak ribuan mil dari Perancis menuju ke kota Makah untukmelaksanakan haji hanya dengan mengendarai sebuah mobil tua. Penelitian inibertujuan untuk mengetahui pesan moral islami apa saja yang terkandung dalamfilm Le grand voyage dan bagaimana pesan tersebut dimunculkan dalam film.Penelitian ini menggunakan teori Struktural untuk menjawab rumusan masalah.Penelitian ini merupakan penelitian kualitatif dengan menggunakan teknik studipustaka serta dokumentasi sebagai metode pengumpulan data, dan teknikdeskriptif dalam proses analisis data.Berdasarkan hasil penelitian ini, terdapat 13 pesan moral islami yang terkandungdalam film Le grand voyage. Semua pesan moral tersebut mengacu pada sebuahproses perbaikan moral dan spiritual antara manusia dengan manusia dan alam,serta antara manusia dengan TuhanPenulis menyarankan pada penelitian selanjutnya untuk meneliti film Le grandvoyage menggunakan pendekatan sosiologi sastra yang nantinya dapat mengupashal apa saja yang melatarbelakangi pembuatan film ini dan tujuan sebenarnyayang ingin dicapai oleh pembuat film Le grand voyage

The Consumer Quality Index Hip Knee Questionnaire measuring patients' experiences with quality of care after a total hip or knee arthroplasty

<p>Abstract</p> <p>Background</p> <p>The Dutch Consumer Quality Index Hip Knee Questionnaire (CQI Hip Knee) was used to assess patients' experiences with and evaluations of quality of care after a total hip (THA) or total knee arthroplasty (TKA). The aim of this study is to evaluate the construct validity and internal consistency reliability of this new instrument and to assess its ability to measure differences in quality of care between hospitals.</p> <p>Methods</p> <p>Survey data of 1,675 subjects who underwent a THA or TKA were used to evaluate the psychometric properties. Exploratory factor analyses were performed and item-total correlations and inter-factor correlations were calculated to assess the construct validity of the instrument. Reliability analyses included tests of internal consistency (Cronbach's alpha coefficients). Finally, multilevel analyses were performed to assess the ability of the instrument to discriminate between hospitals in quality of care.</p> <p>Results</p> <p>Exploratory factor analyses indicated that the survey consisted of 21 items measuring five aspects of care (i.e. communication with nurses, communication with doctors, communication with general practitioner, communication about new medication, and pain control). Cronbach's alpha coefficients ranged from 0.76 to 0.90 indicating good internal consistency. The survey's ability to discriminate between hospitals was partly supported by multilevel analysis. Two scales (i.e. communication with nurses and communication with doctors) were able to measure differences between hospitals with respect to patients' experiences with quality of care. Logistic multilevel analyses indicated that hospitals explained part of the variation between patients in receiving information.</p> <p>Conclusion</p> <p>These findings suggest that the CQI Hip Knee is reliable and valid for use in Dutch health care. Health care providers or health plans can use this survey to measure patients' experiences with hospital care and to identify variations in care between hospitals.</p

Metabolite Cross-Feeding Enhances Virulence in a Model Polymicrobial Infection

Microbes within polymicrobial infections often display synergistic interactions resulting in enhanced pathogenesis; however, the molecular mechanisms governing these interactions are not well understood. Development of model systems that allow detailed mechanistic studies of polymicrobial synergy is a critical step towards a comprehensive understanding of these infections in vivo. In this study, we used a model polymicrobial infection including the opportunistic pathogen Aggregatibacter actinomycetemcomitans and the commensal Streptococcus gordonii to examine the importance of metabolite cross-feeding for establishing co-culture infections. Our results reveal that co-culture with S. gordonii enhances the pathogenesis of A. actinomycetemcomitans in a murine abscess model of infection. Interestingly, the ability of A. actinomycetemcomitans to utilize L-lactate as an energy source is essential for these co-culture benefits. Surprisingly, inactivation of L-lactate catabolism had no impact on mono-culture growth in vitro and in vivo suggesting that A. actinomycetemcomitans L-lactate catabolism is only critical for establishing co-culture infections. These results demonstrate that metabolite cross-feeding is critical for A. actinomycetemcomitans to persist in a polymicrobial infection with S. gordonii supporting the idea that the metabolic properties of commensal bacteria alter the course of pathogenesis in polymicrobial communities

Characterization of ftsZ Mutations that Render Bacillus subtilis Resistant to MinC

Background: Cell division in Bacillus subtilis occurs precisely at midcell. Positional control of cell division is exerted by two mechanisms: nucleoid occlusion, through Noc, which prevents division through nucleoids, and the Min system, where the combined action of the MinC, D and J proteins prevents formation of the FtsZ ring at cell poles or recently completed division sites. Methodology/Principal Findings: We used a genetic screen to identify mutations in ftsZ that confer resistance to the lethal overexpression of the MinC/MinD division inhibitor. The FtsZ mutants were purified and found to polymerize to a similar or lesser extent as wild type FtsZ, and all mutants displayed reduced GTP hydrolysis activity indicative of a reduced polymerization turnover. We found that even though the mutations conferred in vivo resistance to MinC/D, the purified FtsZ mutants did not display strong resistance to MinC in vitro. Conclusions/Significance: Our results show that in B. subtilis, overproduction of MinC can be countered by mutations that alter FtsZ polymerization dynamics. Even though it would be very likely that the FtsZ mutants found depend on other Z-ring stabilizing proteins such as ZapA, FtsA or SepF, we found this not to be the case. This indicates that the cell division process in B. subtilis is extremely robust.